Numerous products of commercial interest, such as intermediates of L-ascorbic acid, have been produced biocatalytically in genetically engineered host cells. L-Ascorbic acid (vitamin C, ASA) is commonly used in the pharmaceutical and food industries as a vitamin and antioxidant, and due to this relatively large market volume and high value as a specialty chemical the synthesis of ASA has received considerable attention.
A chemical synthesis route from glucose to ASA, commonly known as the Reichstein-Grussner method, was first disclosed in 1934 (Reichstein T. et al., (1934) Helv. Chim. Acta., 17:311-328 and Reichstein T. et al. (1933) Helv. Chim. Acta. 16: 561, 1019). A bioconversion method for the production of an ASA intermediate, 2-KLG, has been disclosed by Lazarus et al. (1989, “Vitamin C: Bioconversion via a Recombinant DNA Approach”, GENETICS AND MOLECULAR BIOLOGY OF INDUSTRIAL MICROORGANISMS, American Society for Microbiology, Washington D.C. Edited by C. L. Hershberger). This bioconversion of a carbon source to KLG involves a variety of intermediates, and the enzymatic process is associated with co-factor dependent 2,5-DKG reductase activity (DKGR). Additionally, recombinant DNA techniques have been used to bioconvert glucose to KLG in Erwinia herbicola in a single fermentative step (Anderson, S. et al., (1985) Science 230:144-149). Effective procedures for converting KLG to ASA are described in Crawford et al., ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, 37:79-155 (1980).
A number of strategies have been explored in bacteria and other microorganisms to increase the production of ASA intermediates. Some of these strategies include; gene deletions, gene additions and random mutagenesis. In particular, some gene manipulation strategies are mentioned below and include: (i) overexpressing particular genes in the ASA pathway (Anderson et al., (1985) Sci. 230:144-149; Grindley et al., (1988) Appl. Environ. Microbiol. 54: 1770; and U.S. Pat. No. 5,376,544); (ii) mutating genes encoding glycolytic enzymes (Harrod, et al. (1997) J. Ind. Microbiol. Biotechnol. 18:379-383; Wedlock, et al. (1989) J. Gen. Microbiol. 135: 2013-2018; and Walsh et al. (1983) J. Bacteriol. 154:1002-1004); (iii) utilizing bacterial host strains deficient in glucokinase (Japanese patent publication JP 4267860; Russell et al. (1989) Appl. Environ. Microbiol. 55: 294-297; Barredo et al. (1988) Antimicrob. Agents-Chemother 32: 1061-1067; and DiMarco et al. (1985) Appl. Environ. Microbiol. 49:151-157); (iv) reducing metabolism of glucose or gluconate by deletion of a gene required for phosphorylation, for example deleting the glucokinase gene (glkA) or gluconokinase gene (gntK) (WO 02/081440); and (v) reducing metabolism by manipulating enzymes involved in carbon utilization downstream of the initial glucose phosphorylation, for example manipulating phosphoglucose isomerase, phosphofructokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase or 6-phosphogluconate dehydratase.
Despite the above strategies, problems still persist concerning the diversion of ASA intermediates for catabolic metabolic purposes, and this results in reducing the efficiency and overall production of ASA intermediates. Thus, there still remains a need for improved production methods for ASA intermediates which are coupled to the metabolic pathways of host cells.
Carbon sources, such as glucose and gluconate, involved in the production of ASA intermediates may be separated by a cellular membrane from the reactions which utilize these substrates. When such a substrate and the synthetic machinery are separated, production of the product may require translocation of the substrate to the site of the synthetic reaction. Alternatively products generated inside the cell may require translocation from within the cell. Therefore, altering a substrate transport system may result in increased or decreased substrate availability for a particular metabolic pathway.
The present invention provides altered bacterial strains which include non-functional gluconate transporter molecules. The altered bacterial strains, which include the non-functional transporter molecules, have an increased amount of carbon substrate, such as glucose that may be utilized for the production of desired products, such as ASA intermediates.